Characterization of the Products of the Heme Detoxification Pathway in Malarial Late Trophozoites by X-ray Diffraction

Abstract

In a process inhibited by the quinoline antimalarial drugs, Plasmodia detoxify heme released during the degradation of hemoglobin by aggregating it into malarial pigment, an insoluble crystalline heme coordination polymer. Synchrotron x-ray powder diffraction patterns for intact desiccated malarial trophozoites and synthetic ␤-hematin have been measured; both materials correspond to a single crystalline triclinic lattice with unit cell parameters a ‫؍‬ 12.2176(4), b ‫؍‬ 14.7184(5), c ‫؍‬ 8.0456(3) Å; ␣ ‫؍‬ 90.200(2), ␤ ‫؍‬ 96.806(3), ␥ ‫؍‬ 97.818(3)°a nd Z ‫؍‬ 2. These results unambiguously demonstrate that hemozoin crystallites are identical to synthetic ␤-hematin.Heme is a potent multifunction regulator whose biochemical levels and distribution are precisely controlled on both intraand extracellular levels (1). Efficient regulation of heme is particularly critical for intra-erythrocytic parasites such as plasmodia which process large quantities of heme in the postinvasion digestion of the erythrocyte's hemoglobin. Plasmodia, which lack heme oxygenases, detoxify heme by sequestering it into an insoluble heme aggregate termed malarial pigment or hemozoin (2). The quinoline-based family of antimalarials interfere with this process by an as yet unknown mechanism that has recently come under intense scrutiny as part of the effort to combat the spread of chloroquine-resistant strains of Plasmodium. A variety of spectroscopic and bioanalytical techniques indicate that hemozoin is similar to the synthetic aggregated heme phase ␤-hematin, which is thought to form strands of hemes linked by propionate oxygen-iron bonds as well as interstrand propionate hydrogen bonds, Fig. 1 (3-5). Characterization of the carboxylate stretching bands for the propionic acid side chains by IR and Raman spectroscopy provides the best evidence for the presence of iron-oxygen bonds to the propionate side chains (3-5). Unfortunately, crystallographic characterization of these heme aggregates has been hampered by the phase heterogeneity of many synthetic preparations as well as by the small size of the synthetic and natural crystallites isolated from either trophozoites and infected hosts (6). High resolution powder diffraction has been used extensively for the solution of many structural problems (7), and it can solve problems posed by diffraction from microcrystalline phases. In this communication we describe the characterization of ␤-hematin derived from both synthetic and natural sources and provide new unambiguous evidence that the heme aggregate present in late stage trophozoites is ␤-hematin. EXPERIMENTAL PROCEDURESCirca 3 ϫ 10 9 chloroquine-susceptible Plasmodium falciparum late trophozoites of the 3D7 clone (NF54 strain) were synchronized with sorbitol and allowed to achieve a 92% level of parasitemia (8). The resulting intact trophozites were isolated by rapid cooling in liquid nitrogen, and the frozen suspension of cells was lyophilized by freezedrying in vacuo at Ϫ10°C. In general this freeze-drying process required 12-14 h and res...

Macrophage Preconditioning with Synthetic Malaria Pigment Reduces Cytokine Production via Heme Iron-Dependent Oxidative Stress

“…The study of such interactions is simplified now by the availability of ␤-hematin (BH), a synthetic polymer obtained from hematin in acidic conditions. Synthetic pigment is identical to the heme portion of native hemozoin, as conclusively demonstrated by experiments using X-ray diffraction and Fourier transforminfrared (FT-IR) spectroscopy (Bohle et al, 1997;Pagola et al, 2000;Slater et al, 1991).…”

“…This study may help correlate the different clinical manifestations of malaria, ranging from uncomplicated to severe disease, with dysregulation of phagocyte functions and promote better therapeutic strategies to counteract the effects of hemozoin accumulation. (Lab Invest 2000, 80:1781-1788.I ntraerythrocytic stage plasmodia digest hemoglobin in the food vacuole, where the globin is degraded and heme is detoxified, principally by polymerization/crystallization into hemozoin, an insoluble, microcrystalline derivative of ferri-protoporphyrin IX (␣ hematin, AH) (Bohle et al, 1997;Francis et al, 1997;Pagola et al, 2000;Slater et al, 1991). As parasites mature, hemozoin (malaria pigment) accumulates in the food vacuole and remains in the "residual body" after schizont rupture.…”

Macrophage populations of different origins have distinct susceptibilities to lipid peroxidation induced by β‐haematin (malaria pigment)

“…Repeated cycles of phagocytosis as well as the production of oxygen radicals and nitrogen oxides are inhibited [7,8]. In addition, we also showed that peritoneal mouse macrophages fed with [3-haematin, a synthetic polymer of Fe(III)-PPIX identical to native haemozoin [9,10] failed to produce TNFcx and Nitric Oxide (NO) when stimulated by lipopolysaccharide (LPS) [11]. Two aspects of this phenomenon are worth noting: (i) by contrast to peritoneal macrophages, microglia cells derived fi'om the same mouse strain proved quite refractory to the [3-haematininduced functional inhibition; (ii) the inhibition of NO and TNFc~ production observed in peritoneal macrophages was partially counterbalanced by the addition of sulphydryl group donors such as 2-mercaptoethanol, N-acetylcysteine and glu-*Corresponding author.…”

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

“…Hence, an understanding of the mechanisms of hematin crystallization and its inhibition by antimalarials may prove to be influential for drug development (14). Despite many years of effort (7,9,12,(15)(16)(17)(18), fundamental questions regarding the mechanism of hematin crystallization and its inhibition remain elusive. Among them are (i) What is the nature of the environment within the parasite where hemozoin crystals recruit hematin and grow?…”